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Efficient One-Step Synthesis of Biologically Related Lariat RNAs by a Deoxyribozyme.

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Angewandte
Chemie
Figure 1. Lariat RNA and its synthesis by a deoxyribozyme (B = A, G,
C, or U). a) Connectivity of lariat RNA and 2’,5’-branched RNA. The
latter has the same branch-site nucleotide as a lariat but lacks the
closed loop. The four illustrated nucleotides constitute the minimal
part of a 2’,5’ -branched RNA. b) Deoxyribozyme-catalyzed synthesis of
lariat RNA.
DNA Catalysis
DOI: 10.1002/ange.200501643
Efficient One-Step Synthesis of Biologically
Related Lariat RNAs by a Deoxyribozyme**
Yangming Wang and Scott K. Silverman*
Lariat RNAs are intermediates in RNA splicing events
catalyzed by group II introns and the spliceosome.[1, 2] Lariats
have a closed RNA loop with a single 2’–5’ linkage and a
single-stranded oligonucleotide attached at the 3’-oxygen
atom of the branch-site nucleotide (Figure 1 a). Topologically,
[*] Y. Wang, Prof. S. K. Silverman
Department of Chemistry
University of Illinois at Urbana-Champaign
600 South Mathews Avenue, Urbana, IL 61801 (USA)
Fax: (+ 1) 217-244-8024
E-mail: scott@scs.uiuc.edu
[**] This research was supported by the Burroughs Wellcome Fund
(New Investigator Award in the Basic Pharmacological Sciences),
the March of Dimes Birth Defects Foundation (Research Grant No.
5-FY02-271), the National Institutes of Health (GM-65966), the
American Chemical Society Petroleum Research Fund (38803-G4),
and the UIUC Department of Chemistry (all to S.K.S.). S.K.S. is a
Fellow of The David and Lucile Packard Foundation. We thank
Jonathan Staley (University of Chicago) for the plasmid encoding
the ACT1 intron RNA and Scott Stevens (University of Texas at
Austin) for the sample of debranching enzyme.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. 2005, 117, 6013 –6016
lariats are a subclass of 2’,5’-branched RNAs, which do not
necessarily have the closed loop that is characteristic of
lariats. Owing to their special topology, lariat RNAs are
difficult to synthesize by any conventional chemical approach
such as solid-phase synthesis,[3] and even the simpler 2’,5’branched RNA core (without the closed loop of a lariat) is a
significant challenge.[4] Recently we reported artificial deoxyribozymes (DNA enzymes)[5] that create branched RNAs in
> 90 % yield by catalyzing the intermolecular reaction of an
internal 2’-hydroxy group with a 5’-triphosphate.[6, 7] Some of
these deoxyribozymes are capable of synthesizing branched
RNAs of wide sequence composition.[8, 9] Lariat products can
result from intramolecular branch formation events (that is,
macrocyclizations with single linear RNA substrates), as
illustrated in Figure 1 b. Apart from using the frequently
impractical biological splicing machinery itself, there is no
method present for the synthesis of biologically relevant lariat
RNAs, which are often several hundred nucleotides in length
and have extensive secondary structure[10] that may interfere
with loop formation. Indeed, most of our reported branchforming deoxyribozymes such as 7S11, which is widely useful
for branched RNA synthesis,[8] are not useful with biologically
derived RNA sequences as substrates (lariat yield typically
< 1 %; data not shown). In contrast to these difficulties, we
report herein that the 6BX22 deoxyribozyme can create two
common classes of biological lariat RNAs efficiently in one
step from readily available RNA substrates.
In previous efforts, we used in vitro selection[11] to identify
many deoxyribozymes that synthesize branched RNA.[6–9, 12]
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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However, our procedure does not select for the particular
ability to create lariats. One-step lariat formation is presumably more difficult than simple branch formation because the
incipient RNA loop may clash sterically with the DNA
structure, thereby inhibiting catalysis. We surveyed many of
the branch-forming deoxyribozymes to determine their lariatformation capabilities. One of these deoxyribozymes,
6BX22,[12] showed promise in this regard and was examined
more carefully. The 6BX22 deoxyribozyme has a specific 39nucleotide DNA enzyme region embedded between Watson–
Crick binding arms, as shown schematically in Figure 1 b. It
was found to require Mn2+; detectable RNA ligation activity
was not observed with Mg2+, Ca2+, Zn2+, Fe2+, Co2+, Ni2+,
Cd2+, or [Co(NH3)6]3+ (all as chloride salts at concentrations
in the range of 10 mm–10 mm ; data not shown). The kobs value
for 6BX22-catalyzed branch formation is 0.07 min1 (t1/2
10 min) under standard incubation conditions (Mn2+
(20 mm), pH 7.5, 37 8C; see below). The background rate for
the analogous DNA-templated reaction (the use of a DNA
template that lacks an enzyme region between the DNA
binding arms) is much lower; ktemplated 4 @ 107 min1 [6] ,
which gives a rate enhancement (kobs/ktemplated) of 2 @ 105.[13]
Therefore, 6BX22 clearly “catalyzes” branch formation. If it
merely templated the reaction by increasing the effective
substrate concentration, then kobs/ktemplated would be 1.[5a]
Before studying 6BX22-catalyzed lariat RNA synthesis in
detail, we examined the substrate sequence generality of this
deoxyribozyme for branch formation. This reaction is simpler
than lariat formation because loop closure is not required.
The selection procedure in which 6BX22 was identified[12]
used RNA substrates that correspond to the conserved
branch-site sequences of yeast spliceosomal substrates (Figure 2 a), which are common models for understanding RNA
splicing.[2] Systematic experiments revealed that sequence
changes outside the conserved RNA elements are tolerated
well by 6BX22, indicating that this DNA enzyme is general
for branch formation with yeast spliceosomal substrates
(Figure 2 b). Most nucleotide changes within the conserved
regions are also tolerated. For example, the branch-site
nucleotide itself may be changed from A to C or U with
nearly equivalent ligation efficiencies (Figure 2 c). A branchsite G is accepted but with diminished yield.
We applied 6BX22 toward the synthesis of three specific
representative biological lariat RNAs that are derived from
yeast or mammalian spliceosomal substrates, but share no
sequence elements outside of the small consensus region. The
three RNAs are the 69-nt yeast YBL059W intron with a 51-nt
lariat loop,[14] the 130-nt human b-globin IVS1 intron with a
94-nt loop,[15] and the 309-nt yeast actin (ACT1) intron with a
266-nt loop.[16] In each case, a linear 5’-triphosphate substrate
was prepared by in vitro transcription with T7 RNA polymerase. By using the linear substrate, the small YBL059W
lariat was readily synthesized by 6BX22 in one step and in
high yield (Figure 3 a). The larger b-globin and ACT1 lariats
were also readily prepared (Figure 3 b,c). The lariats were
formed with kobs values similar to those of the analogous
branches, or in the case of b-globin, about sixfold slower (but
still with a kobs value that is preparatively useful). Compelling
evidence for each lariat structure was provided by several
6014
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Figure 2. Branched RNA formation by the 6BX22 deoxyribozyme.
a) With yeast spliceosomal substrate sequences; the conserved nucleotides are shown explicitly.[2] As demonstrated with the comprehensive
experiments shown herein and in the Supporting Information, 6BX22
tolerates nucleotide changes at most of the conserved positions; the
sites of tolerance are indicated with uppercase letters. The second nucleotide (U) of the right-hand substrate (R) prefers U, C, or A over G.
The sequence of the enzyme region of 6BX22 (the 39 nucleotides not
base-paired with the RNA substrates) is given below the structure. The
Watson–Crick binding arms of the DNA are shown in light gray. See
Experimental Section for full sequences of the left-hand (L) and righthand (R) substrates. b) Demonstration of the generality of 6BX22 for
its RNA substrate sequences; conditions: HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulfonic acid), 50 mm, pH 7.5), NaCl (150 mm),
KCl (2 mm), MnCl2 (20 mm), 37 8C. Original RNA sequences that correspond to the core nucleotides of the ACT1 RNA (~); variant RNA
sequences (*, see Experimental Section). For this experiment, kobs =
0.077 0.007 min1 (original sequences) and 0.066 0.004 min1
(variant sequences); errors are standard deviations from exponential
curve fits. c) Demonstration of the generality of 6BX22 for branch-site
nucleotides (t = 0, 0.5, and 1.5 h with 5’-32P-radiolabeled L substrate;
20 % PAGE). L + R product yields at t = 1.5 h: branch-site A, 71 %; G,
5 %; C, 33 %; U, 64 % (krel = 1, 0.085, 0.097, and 0.027, respectively;
data not shown). In all cases, partial alkaline hydrolysis of the
branched product[6] verified that the site of branching remained
unchanged (data not shown).
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. 2005, 117, 6013 –6016
Angewandte
Chemie
Figure 3. Formation of biologically related lariat RNAs with the 6BX22
deoxyribozyme. a) Synthesis of the 69-nt YBL059W lariat, which has a
51-nt loop (12 % PAGE; t = 0, 0.5, and 1.5 h). Yield at t = 1.5 h: 52 %;
kobs = 1.1 h1 (data not shown). b) Synthesis of the 130-nt human bglobin IVS1 lariat, which has a 94-nt loop (8 % PAGE; t = 0, 2, and
6 h). Yield at t = 6 h: 33 %; kobs = 0.17 h1. At t = 24 h the yield was
50 %, but some nonspecific RNA degradation was evident (data not
shown). c) Synthesis of the 309-nt ACT1 lariat, which has a 266-nt
loop; this was tested with mutants in which the branch-site nucleotide
was changed as indicated (6 % PAGE; t = 0, 0.5, and 1.5 h). Yields at
t = 1.5 h: branch-site A, 72 %; G, 9 %; C, 48 %; U, 70 %. For branchsite A kobs = 2.8 h1 (data not shown). For the b-globin lariat, disruptor
DNA oligonucleotides were required to sequester RNA secondary
structure and enable binding of the DNA enzyme to the RNA substrates (Supporting Information). Without disruptors, the b-globin
lariat yield was 0.3 % at 6 h. A disruptor oligonucleotide was helpful
but not absolutely required for ACT1 lariat synthesis, primarily by
modestly enhancing the ligation rate (by less than twofold) and by
suppressing nonspecific RNA degradation.
biochemical assays in which the lariat RNA was cleaved with
the 10–23 deoxyribozyme[17] and yeast debranching enzyme[18]
to generate the predicted pattern of gel bands (Supporting
Information). For the b-globin lariat, disruptor DNA oligonucleotides were required to allow the deoxyribozyme to bind
productively with its RNA substrates (Supporting Information). For the largest ACT1 lariat, a disruptor helped to
prevent RNA degradation, but it was not required for high
lariat yield (data not shown).
The yeast ACT1 intron is a particularly common model
system for studying spliceosomal RNA processing.[16, 19] Artificial synthesis of such lariats without the natural splicing
machinery will enable many biochemical experiments,
because the sequence requirements of the spliceosome need
not be obeyed. For example, the tolerance of 6BX22 toward
changes of the branch-site nucleotide during branch formation (Figure 2 c) is maintained for lariat synthesis (Figure 3 c).
Therefore, for the first time, biochemists have access to “real”
spliceosomal lariats with mutations at their key branch-site
nucleotides. To enable such experiments, preparative-scale
(nanomole) synthesis of the ACT1 intron lariat was successfully carried out in high yield (Figure 4).
In summary, we have demonstrated that the 6BX22
deoxyribozyme catalyzes efficient and general one-step
Angew. Chem. 2005, 117, 6013 –6016
Figure 4. Preparative synthesis of the ACT1 lariat RNA; the image was
recorded by UV shadowing of the 6 % polyacrylamide gel.
lariat synthesis with biological RNAs that are derived from
commonly studied yeast and mammalian spliceosomal substrates. The resulting lariats are essentially impossible to
synthesize by traditional organic and solid-phase synthesis
methods. The only available approach until now has required
the use of natural splicing enzymes, which have stringent
sequence requirements that impose considerable limitations
on the RNA sequences that may be used as substrates. Lariat
RNA formation for the YBL059W, b-globin, and ACT1
intron substrates corresponds to the creation of rings with 307,
565, and 1597 atoms, respectively. These DNA-catalyzed
macrocyclization reactions succeed without the use of protecting groups despite the presence of hundreds of competitive 2’-hydroxy nucleophiles. This demonstrates an extremely
high level of site-selectivity. The reactions also produce very
small amounts of side products such as RNA substrate
oligomers that are created in large quantity with other
deoxyribozymes that synthesize lariat RNAs in low yield.[6]
The broadly useful lariat synthesis capacity of 6BX22 is
unique in comparison with all of our other deoxyribozymes
identified so far, including those such as 7S11, which are quite
general for the formation of simpler branched RNAs.[8] The
structural basis by which 6BX22—yet none of our other
deoxyribozymes—readily tolerates a closed RNA loop in its
catalytically active conformation requires further study, as
does the mechanism by which 6BX22 achieves its > 105-fold
rate enhancement over mere templating. We continue to
investigate these fundamental features of catalytic DNA, and
we are using synthetic lariats created by 6BX22 to examine
key biochemical aspects of RNA splicing.
Experimental Section
Systematic variation of RNA substrate sequences (Figure 2): The
original left-hand (L) RNA substrate sequence (which corresponds to
that used in the selection procedure[12]) was 5’-GGAAGUCUCAUGUACUAACA-3’. The original right-hand (R) RNA substrate
sequence was 5’-GUAUGUUCUAGCGCGGA-3’. Together these
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Zuschriften
sequences comprise the “core” of the ACT1 branch. For the
experiment shown in Figure 2 b, nearly all RNA substrate nucleotides
in both L and R were changed by transversions (A$C and G$U). In
L, changes were made to all nucleotides from the 5’ end through and
including UACU. In R, changes were made to all nucleotides after 5’GU through the 3’ end. In all cases, corresponding transversions were
made to the DNA to maintain Watson–Crick complementarity. For
the experiment shown in Figure 2 c, the L substrate was varied only at
the branch-site nucleotide position (UACUAAC). (For experiments
with variations at the L substrate UACUA nucleotide and at the R
substrate GU nucleotide, see the Supporting Information.) Variation
of the 5’-triphosphorylated guanosine (5’-pppG) of the R substrate
was not tested. The assays were performed with 5’-32P-radiolabeled L
substrate and L/deoxyribozyme/R = 1:3:6, under incubation conditions of HEPES (50 mm, pH 7.5), NaCl (150 mm), KCl (2 mm), and
MnCl2 (20 mm) at 37 8C.
Synthesis of the three lariat RNAs (Figure 3): Each linear RNA
substrate was prepared by transcription in the presence of a-32P-CTP
to give internally 32P-radiolabeled transcript. The assays were carried
out under the incubation conditions above and a substrate/deoxyribozyme ratio of 1:2. In all cases, side products (presumably substrate
oligomers[6]) were observed in very small amounts: < 3 % yield for
YBL059W and b-globin; < 0.1 % yield for ACT1 (data not shown).
Preparative ACT1 lariat synthesis (Figure 4): A sample was
prepared that contained 1.0 nmol linear substrate and 1.5 nmol
deoxyribozyme plus 2.0 nmol disruptor DNA oligonucleotide (Supporting Information) in HEPES (5 mm, pH 7.5), NaCl (15 mm), and
EDTA (ethylenediaminetetraacetic acid, 0.1 mm) in a volume of
150 mL. The sample was annealed by heating at 95 8C for 3 min and
then cooling on ice for 5 min. The volume was increased to 200 mL:
HEPES (50 mm, pH 7.5), NaCl (150 mm), KCl (2 mm), and MnCl2
(20 mm) ; Mn2+ was added from an aqueous stock solution (1m). The
200-mL solution was incubated at 37 8C for 1.5 h and then mixed with
300 mL low-dye stop solution (80 % formamide, 1 @ TBE (tris(hydroxymethyl)aminomethane and boric acid, 89 mm each, pH 8.3), and
xylene cyanol and bromophenol blue, 0.0025 % each). The sample
was resolved by 6 % PAGE and visualized by UV shadowing; the
lariat product was extracted and ethanol-precipitated as described.[7]
The isolated yield of lariat RNA product (after gel extraction and
ethanol precipitation) was 0.39 nmol starting from 1.0 nmol of linear
substrate. This 39 % yield (compared with the 70 % yield observed at
the analytical scale without PAGE purification; Figure 3 c) reflects
losses that are commonly observed during extraction and precipitation of large RNA species from polyacrylamide gels.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
Received: May 12, 2005
Published online: August 5, 2005
.
Keywords: deoxyribozymes · DNA · enzyme catalysis ·
lariat RNA · macrocycles
[19]
[20]
Rasmussen, G. Brandenburg, J. Wengel, Tetrahedron 1995, 51,
8491; d) M. Grøtli, R. Eritja, B. Sproat, Tetrahedron 1997, 53,
11 317.
a) S. K. Silverman, Org. Biomol. Chem. 2004, 2, 2701; b) J. C.
Achenbach, W. Chiuman, R. P. Cruz, Y. Li, Curr. Pharm.
Biotechnol. 2004, 5, 321; c) G. M. Emilsson, R. R. Breaker,
Cell. Mol. Life Sci. 2002, 59, 596; d) Y. Lu, Chem. Eur. J. 2002, 8,
4589.
Y. Wang, S. K. Silverman, J. Am. Chem. Soc. 2003, 125, 6880.
Y. Wang, S. K. Silverman, Biochemistry 2003, 42, 15 252.
a) R. L. Coppins, S. K. Silverman, Nat. Struct. Mol. Biol. 2004,
11, 270; b) R. L. Coppins, S. K. Silverman, J. Am. Chem. Soc.
2005, 127, 2900.
E. D. Pratico, Y. Wang, S. K. Silverman, Nucleic Acids Res. 2005,
33, 3503.
For example, see the Supporting Information for secondary
structures of two of the RNAs studied for the work reported
herein.
a) G. F. Joyce, Annu. Rev. Biochem. 2004, 73, 791; b) D. S.
Wilson, J. W. Szostak, Annu. Rev. Biochem. 1999, 68, 611.
Y. Wang, S. K. Silverman, Biochemistry 2005, 44, 3017.
If one or more (e.g., 39) random DNA nucleotides are included
between the DNA-binding arms in place of the enzyme region,
then ktemplated decreases considerably (data not shown). Therefore, the 6BX22 rate enhancement calculated by using ktemplated
for the DNA template that lacks any enzyme region is a
conservative (lower-limit) estimate. Furthermore, as the templated background product is almost certainly linear and not
branched RNA[20] (insufficient amounts of this product were
available for analysis), the ktemplated value for the formation of the
branched product that 6BX22 actually synthesizes must be lower
than the experimentally measured ktemplated value. This lends
further credence to the calculated rate enhancement as a lowerlimit estimate.
a) C. A. Davis, L. Grate, M. Spingola, M. Ares, Jr., Nucleic Acids
Res. 2000, 28, 1700; b) L. Grate, M. Ares, Jr., Methods Enzymol.
2002, 350, 380.
a) R. A. Spritz, P. Jagadeeswaran, P. V. Choudary, P. A. Biro, J. T.
Elder, J. K. deRiel, J. L. Manley, M. L. Gefter, B. G. Forget, S. M.
Weissman, Proc. Natl. Acad. Sci. USA 1981, 78, 2455; b) R.
Reed, T. Maniatis, Cell 1985, 41, 95; c) K. B. Hall, M. R. Green,
A. G. Redfield, Proc. Natl. Acad. Sci. USA 1988, 85, 704.
R. Ng, J. Abelson, Proc. Natl. Acad. Sci. USA 1980, 77, 3912.
S. W. Santoro, G. F. Joyce, Proc. Natl. Acad. Sci. USA 1997, 94,
4262.
a) S. L. Ooi, C. Dann, 3rd, K. Nam, D. J. Leahy, M. J. Damha,
J. D. Boeke, Methods Enzymol. 2001, 342, 233; b) K. Nam, R. H.
Hudson, K. B. Chapman, K. Ganeshan, M. J. Damha, J. D.
Boeke, J. Biol. Chem. 1994, 269, 20 613.
J. P. Staley, C. Guthrie, Mol. Cell 1999, 3, 55.
R. Rohatgi, D. P. Bartel, J. W. Szostak, J. Am. Chem. Soc. 1996,
118, 3340.
[1] C. L. Peebles, P. S. Perlman, K. L. Mecklenburg, M. L. Petrillo,
J. H. Tabor, K. A. Jarrell, H. L. Cheng, Cell 1986, 44, 213.
[2] C. B. Burge, T. Tuschl, P. A. Sharp in The RNA World, 2nd ed.
(Eds.: R. F. Gesteland, T. R. Cech, J. F. Atkins), Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, NY, 1999,
pp. 525 – 560.
[3] a) C. Sund, P. Agback, J. Chattopadhyaya, Tetrahedron 1991, 47,
9659; b) C. Sund, P. Agback, J. Chattopadhyaya, Tetrahedron
1993, 49, 649; c) C. B. Reese, Q. Song, Nucleic Acids Res. 1999,
27, 2672; d) S. Carriero, M. J. Damha, J. Org. Chem. 2003, 68,
8328.
[4] a) M. J. Damha, K. Ganeshan, R. H. Hudson, S. V. Zabarylo,
Nucleic Acids Res. 1992, 20, 6565; b) B. S. Sproat, B. Beijer, M.
Grøtli, U. Ryder, K. L. Morand, A. I. Lamond, J. Chem. Soc.
Perkin Trans. 1 1994, 419; c) M. von BPren, G. V. Petersen, K.
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